Multidrug resistances pose a major obstacle to the effective treatment of many severe human diseases and remain an enormous public health problem because they are responsible for the loss of effectiveness of many anticancer and antiviral agents. High resolution structures of bacterial ABC transporters that are homologous to multidrug resistance proteins have been published. Recently the first X-ray structure of a eukaryotic multidrug resistance protein, P-glycoprotein (P-gp), that is thought to confer drug resistance in cancers and HIV infection, has been reported. Our group has used these structural models as well as homology models based on conserved structures to investigate the mechanism of drug export. Computational drug docking studies have revealed putative drug binding sites on both the human and Sav1866 pumps that correlate well with previously reported biochemical results. Most of the drug binding sites were observed in the transmembrane helical part of the protein, potentially making these sites part of the ATP-driven drug expulsion pathway of the enzyme. We hypothesize that inhibitors of P-gp that would chemosensitize drug resistant cells will be most efficient if they interact with the nucleotide binding sites of the enzyme and inhibit the energy harvesting steps of catalysis. In Aim 1 of this proposed study we will screen at least 9.5 million members of a drug-like chemical database for molecules that bind with high calculated affinity to the nucleotide binding domains, but that bind weakly at drug binding domains of P-gp. Candidates with these properties are hypothesized to inhibit P-gp, while not being good transport substrates. In Aim 2 we will test the small molecules identified in Aim 1 for the ability to specifically inhibit ATP- hydrolysis of purified, soluble P-glycoprotein as well as drug-stimulated ATP-hydrolysis of P-gp without negatively affecting other important ATP-utilizing enzymes. We will determine if binding of the inhibitors changes the overall affinity of P-gp for ATP in ESR studies using spin-labeled ATP. We will also determine whether the inhibitor molecules change the way that the enzyme hydrolyzes ATP by comparing transition state characteristics in the absence or presence of drug with and without normal transport substrate present. These studies are expected to provide us with a set of drug-like molecule leads that may translate into drugs to be co-administered with chemotherapeutics for effective chemotherapy. At the very least the identified molecules will serve as good candidates for rational drug design for these much needed inhibitors of multidrug resistance proteins. PUBLIC HEALTH RELEVANCE: Multidrug resistance phenomena remain an enormous public health problem and a major obstacle to the effective treatment of many severe human diseases. Up to ~40% of all human cancers develop multidrug resistance. These phenomena are responsible for the loss of effectiveness of many anticancer and antiviral agents and are root cause of many of the emerging antibiotic resistances of microorganisms. Even though considerable effort has been expended in the elucidation of the structure and enzymatic mechanism of the family of proteins responsible for multidrug resistance, there remain large gaps in our understanding of these important enzymes. The discovery and development of effective drugs that inhibit these enzymes and allow effective treatment of often intractable diseases like cancer or HIV infection will be of great importance. In this project we propose to in-silico screen large chemical data bases for small molecules that specifically and tightly interact with the energy harvesting parts of the multidrug resistance P-glycoprotein. Good candidates will then be tested in the lab as to their inhibitory effect on ATP hydrolysis and ATP binding to the enzyme, the steps thought to be needed for effective drug export. Drug candidates that are shown in the lab to inhibit these steps will be tested to extrapolate their potential effects on other important nucleotide binding proteins. Such small molecules that strongly inhibit P-gp activity while not or weakly interacting with other classes of enzymes may then be further developed for co-therapeutics in therapy of drug resistant cancers or HIV patients.